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Although hematological disorders with salient features of thrombocytopenia have been well documented in dengue patients, the role of CD61-expressing platelets and the megakaryocytic cell lineage in the pathogenesis of dengue virus (DENV) infection remains largely unexplored. A prospective observational study was performed using blood samples and PBMCs from dengue-confirmed patients, as well as from rhesus monkeys (RM) experimentally infected with DENV. Immunohistochemical staining and FACS techniques were applied to evaluate the frequencies of CD61(+) cells that contained DENV antigen. Highly enriched population of CD61(+) cells was also isolated from acute DENV-infected RM and assayed for DENV RNA by quantitative RT-PCR. Results revealed that DENV antigen was found in small vesicles of varying size, and more frequently in anucleated cells associated with platelets in dengue patients. The DENV antigen-containing cells were CD61(+) and appeared to share characteristics of megakaryocytes. Kinetic profiles of CD61(+) cells from DENV-infected RM revealed a transient increase in CD61(+)CD62P(+) cells early after DENV infection. DENV RNA in a highly enriched population of CD61(+) cells from the infected RM was observed during acute stage. Our results indicate that virus containing CD61(+) cells may be directly linked to the platelet dysfunction and low platelet count characteristics of dengue patients. [ABSTRACT FROM AUTHOR]

Ablative fractional laser-assisted drug delivery has gained attention as a promising method for enhancing dermal drug absorption and improving therapeutic outcomes in dermatological conditions, particularly for hypertrophic and keloid scars. However, despite the growing number of clinical trials and case reports supporting its efficacy, there remains a scarcity of robust evidence on the topical bioavailability and dermato-pharmacokinetics of drugs in human subjects. This study aimed to examine the enhancement of triamcinolone acetonide (TAC) bioavailability following treatment with a fractional Erbium-Doped Yttrium Aluminum Garnet (Er: YAG) laser. Stratum corneum (SC) uptake and transport of TAC from 0.1% TAC cream and 10 mg/mL TAC solution/suspension with and without the laser pre-treatment were determined through tape stripping method for SC collection. TAC therein was quantified by an ultra-performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection. TAC from both formulations without laser assistance was percutaneously absorbed within 6 h and TAC was delivered out from the solution to the SC remarkably higher. When the skin was pre-treated with the laser, permeability of TAC from the solution was escalated by 5 folds. TAC distribution profiles in the SC also confirmed this increased drug uptake, mainly the outer skin layers. On the other hand, amounts of absorbed TAC and their distribution patterns from the cream remained unchanged and low. No adverse events and unbearable pain were observed throughout the experiments. The fractional Er: YAG laser enhanced the dermal absorption of TAC, but this effect was confined to the solution formulation, with no significant improvement seen in the cream. This finding highlights the critical role that drug formulation plays in laser-assisted drug delivery. Moreover, factors such as drug selection, laser type, and optimal laser settings may also impact the efficacy of this approach and require further exploration., Competing Interests: Declarations. Consent for publication: Written informed consent was obtained from the patients for publication of their individual details and accompanying images in this manuscript. The consent form is held by the authors and is available for review by the Editor-in-Chief. Competing interests: The authors declare that they have no conflict of interest. Ethical approval and consent to participate: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable standards. The study was approved by the Institutional Review Board of the Faculty of Medicine Siriraj Hospital at Mahidol University (Si 228/2023). Informed consent was obtained from all individual patients recruited in the study., (© 2024. The Author(s).)

Background: Currently, there is no effective treatment for adult-onset immunodeficiency (AOID) syndrome with anti-interferon-gamma autoantibodies (anti-IFN-γ-auto-Abs). This study aimed to investigate the effectiveness of bortezomib (BTZ) for decreasing anti-IFN-γ-auto-Abs., Methods: A pre- and post-intervention study was conducted from February 2017 through June 2019 at Siriraj Hospital (Bangkok, Thailand). Five patients were invited to receive once-weekly BTZ (1.3 mg/m2 body surface area) subcutaneously for 8 weeks followed by oral cyclophosphamide (1 mg/kg/d) for 4 months. The primary outcomes were the difference in antibody level at 8 and 48 weeks compared with baseline and the incidence of serious adverse events (AEs). The secondary outcome was the occurrence of opportunistic infections (OIs) during the 72 weeks after starting BTZ., Results: The median patient age was 46 years (range, 34-53). All patients had 3-5 OIs prior to enrollment. All patients were receiving antimycobacterial agents for treatment of nontuberculous mycobacterial infection at enrollment. There was no significant difference in the mean optical density of auto-Abs at 8 weeks (3.73 ± 0.72) or 48 weeks (3.74 ± 0.53) compared with baseline (3.84 ± 0.49; P = .336 and P = .555, respectively). However, after serum dilution, the antibody titer nonsignificantly decreased 8-16 weeks after BTZ initiation (P = .345). Ten OIs were observed 24-72 weeks after BTZ initiation., Conclusions: Treatment with BTZ followed by cyclophosphamide yielded no significant decrease in antibody titer levels, and 10 OIs were observed during 24-72 weeks of BTZ treatment. No serious AEs were observed. Combining rituximab with BTZ is likely necessary to prevent generation of new autoantibody-producing plasma cells. Clinical Trials Registration. NCT03103555., Competing Interests: Potential conflicts of interest. N. A. reports honoraria for speaking engagements from AstraZeneca (Thailand), Takeda (Thailand), and Janssen (Thailand). M. P. reports honoraria for speaking engagements from Oxford Nanopore, Rocher (Thailand), and ThermoFisher. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The cell-assisted lipotransfer technique, integrating adipose-derived mesenchymal stem cells (ADMSCs), has transformed lipofilling, enhancing fat graft viability. However, the multipotent nature of ADMSCs poses challenges. To improve safety and graft vitality and to reduce unwanted lineage differentiation, this study refines the methodology by priming ADMSCs into preadipocytes-unipotent, self-renewing cells. We explored the impact of fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF), either alone or in combination, on primary human ADMSCs during the proliferative phase. FGF-2 emerged as a robust stimulator of cell proliferation, preserving stemness markers, especially when combined with EGF. Conversely, FGF-1, while not significantly affecting cell growth, influenced cell morphology, transitioning cells to a rounded shape with reduced CD34 expression. Furthermore, co-priming with FGF-1 and FGF-2 enhanced adipogenic potential, limiting osteogenic and chondrogenic tendencies, and possibly promoting preadipocyte commitment. These preadipocytes exhibited unique features: rounded morphology, reduced CD34, decreased preadipocyte factor 1 (Pref-1), and elevated C/EBPα and PPARγ, alongside sustained stemness markers (CD73, CD90, CD105). Mechanistically, FGF-1 and FGF-2 activated key adipogenic transcription factors-C/EBPα and PPARγ-while inhibiting GATA3 and Notch3, which are adipogenesis inhibitors. These findings hold the potential to advance innovative strategies for ADMSC-mediated lipofilling procedures., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Background: Clerodendrum petasites S. Moore predominantly contains hispidulin (His) and nepetin (Nep) which are immunosuppressive potentials. Although the effect of individual compounds was previously confirmed, a cumulative suppression of these flavonoid mixtures is unknown. This study thus investigated their inhibitory effects and cytotoxicity on T cells by using His:Nep ratios following a naturally occurring dose (3:1) and optimized doses (1:1 and 1:3)., Materials and Methods: Anti-CD3/28 stimulated peripheral blood mononuclear cells were treated with individual compounds and their mixtures. Changes in early cell activation markers in activated T cells and apoptosis were analyzed by a flow cytometer., Results: Mixtures at 3:1 suppressed CD69 and CD25 expression in CD4 + and CD8 + T cells in a dose-dependent manner. At the highest concentration of 200 µM His + 66.7 µM Nep, over 90% inhibition was observed for CD25 expression in CD4 + and CD8 + T cells, whereas a lesser effect was observed for CD69 expression. A dose-dependent inhibition was still observed when using 1:1 and 1:3 ratios. Interestingly, 80-97% inhibition were observed in CD69 and CD25 expression without inducing cell death after treated with the highest doses of each ratio (66.7 µM His + 200 µM Nep and 200 µM His + 200 µM Nep). These mixtures were also exhibited a better suppression than individual compounds., Conclusions: The optimized mixture of His and Nep at 66.7:200 µM is suggested for further study due to a greater suppressive effect than a single compound or a naturally-occurring dose.

Variation in numbers and functions of cells in fat tissues may affect therapeutic outcomes and adverse events after autologous fat tissue grafting in postmastectomy breast cancer patients; however, the relevant information regarding cellular components is still incomplete. Phenotypic characterization of heterogeneous cell subsets in stromal vascular fraction (SVF) isolated from fat tissues by flow cytometry was also limited to a combination of few molecules. This study, therefore, developed a polychromatic staining panel for an in-depth characterization of freshly isolated SVF and expanded adipose-derived stem cells (ADSC) from the patients. ADSC were found predominant in SVF (~65% of CD45 - cells) with a homogenous phenotype of CD13 + CD31 - CD34 + CD45 - CD73 + CD90 + CD105 - CD146 - (~94% of total ADSC). Endothelial progenitor cells (EPC) and pericytes were minor (~18% and ~11% of CD45 - cells, respectively) with large heterogeneity. Downregulation of CD34 and upregulation of CD105 in ADSC were profound at passage 3, showing a phenotype similar to the classical mesenchymal stem cells from the bone marrow. Results from this study demonstrated that fat tissue collected from patients contains ADSC with a highly homogenous phenotype. The in vitro culture of these cells maintained their homogeneity with modified CD34 and CD105 expression, suggesting the expansion from a single population of ADSC., Competing Interests: The authors have no conflicting financial interest., (Copyright © 2020 Premrutai Thitilertdecha et al.)

Burkholderia pseudomallei , the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis., (Copyright © 2020 Jitprasutwit, Jitprasutwit, Hemsley, Onlamoon, Withatanung, Muangsombut, Vattanaviboon, Stevens, Ong, Stevens, Titball and Korbsrisate.)

Background: Hispidulin, nepetin, and vanillic acid are phenolic compounds potentially possessing immunosuppressive property, however, no information on their pharmacological effect and cytotoxicity has been investigated on human T lymphocytes. Materials and methods: Human peripheral blood mononuclear cells were stimulated with anti-CD3/28 coated beads and treated with individual compound at different concentrations (50-200 µM). Inhibition of early cell activation and induction of apoptosis were analyzed by flow cytometric technique. Results: At 200 µM, frequencies of CD25 and CD69 in CD4 + and CD8 + T lymphocytes were markedly decreased by hispidulin and nepetin. When lowering to 100 and 50 µM, hispidulin had no effect on the expression of CD69 in CD4 + T cells, whereas nepetin selectively suppressed CD25 and CD69 expressions in CD8 + T cells at 100 µM and only inhibited CD69 in CD8 + T cells at 50 µM. For vanillic acid, no inhibitory effect was observed while cell activation was significantly increased for all treated concentrations. None of these compounds disturbed levels of total apoptotic cells in CD4 + and CD8 + populations. Conclusions: Hispidulin and nepetin, therefore, exhibit dose-dependent inhibitory activity of early T-cell activation without inducing cell death, considering feasible immunosuppressants for inflammation-related diseases. However, vanillic acid has no effect on immunosuppression but shows more potential on immunostimulation.HighlightsImmunosuppressive effects of hispidulin and nepetin on human T cells were studied.Dose-dependent activity for T-cell suppression was found in hispidulin and nepetin.Vanillic acid showed immunostimulating potential rather than immunosuppression.All compounds did not induce cell death.

CD4 + T cell immunotherapy has potential for treatment in HIV-infected patients. A large number of expanded CD4 + T cells and confirmation of functional-related phenotypes are required for ensuring the successful outcomes of treatment. Freshly isolated CD4 + T cells from healthy donors were activated with anti-CD3/28-coated magnetic beads at different bead-to-cell ratios and cultured in the absence and presence of IL-2 supplementation for 3 weeks. Fold expansion, cell viability, growth kinetic and lymphocyte subset identities were determined. Data demonstrated that a 1:1 bead-to-cell ratio rendered the highest expansion of 1044-fold with 88% viability and 99.5% purity followed by the 2:1 and 0.5:1 ratios. No significant difference in proliferation and phenotypes was found between non-IL-2 and IL-2 supplementation groups. Several specific surface molecule expressions of the expanded cells including chemokine receptors, adhesion molecules, co-stimulatory molecules, activation molecules, maturation markers, cytokine receptors and other molecules were altered when compared to the unexpanded cells. This optimized expansion protocol using the 1:1 bead-to-cell ratio of anti-CD3/28-coated magnetic beads and culture condition without IL-2 supplementation provided the satisfactory yield with good reproducibility. Specific surface molecule expressions of the expanded cells presented potential roles in proliferation, differentiation, homeostasis, apoptosis and organ homing., (© 2019 The Scandinavian Foundation for Immunology.)

Clerodendrum petasites S. Moore has been prescribed in Thai traditional medicine for over 30 years for the treatment of ailments including asthma, inflammation, fever, cough, vomiting, and skin disorders. The phytochemicals from this plant have been identified as phenolic acids, flavones, flavone glycosides, glycosides, phenylpropanoid, and diterpenoid. The pharmacological activities both in vitro and in vivo have mostly been reported from crude extracts and not from pure compounds. This review, therefore, brings together information on the specific phytochemicals found in C. petasites in order to provide a guide to the natural bioactive compounds that are potentially used in medicines together with mechanisms underlying their pharmacological uses. All relevant information was searched for the terms of plant name, naturally-occurring compounds, and traditional uses from reliable databases, such as PubMed, Science Direct and Google Scholar, along with Thai traditional medicine textbooks. There was no specific timeline set for the search and this review selected to report only mechanisms studied by using standard compounds for their biological activities. Four dominant compounds comprising hispidulin, vanillic acid, verbascoside, and apigenin, have robust evidence to support their medical effects. Hispidulin was discovered to be possibly responsible for the treatment of cancer, osteolytic bone diseases, and neurological diseases. Other compounds were also found to tentatively support the uses in inflammation and neurological diseases. C. petasites extracts may provide an option as complimentary medicine, and or for the pharmacological development of new drugs derived from the phytochemicals found within., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Allergen-specific immunotherapy (AIT) facilitates long-term resolution of allergic morbidity resulting in reduced drug use and increased refractoriness to new sensitization. AIT effectiveness has been demonstrated in seasonal and perennial allergies, and insect stings. However, data and studies in AIT relative to cockroach (CR) allergy are relatively scarce. In this study, mice allergic to American CR (Periplaneta americana) were treated with a liposome (L)-entrapped vaccine made of mouse Tregitope289-Per a 9 of the CR, Tregitope167-Per a 9, or Per a 9 alone - or placebo. Allergic mice that received an individual vaccine intranasally had reduced Th2 response, reduced lung inflammation, and reduced respiratory tissue remodeling. However, only L-Tregitope289-Per a 9 and L-Tregitope167-Per a 9 induced expression of immunosuppressive cytokine genes (IL-10, TGF-β, and IL-35 for L-Tregitope289-Per a 9, and IL-10 and TGF-β for L-Tregitope167-Per a 9) and increment of idoleamine-2,3-dioxygenase 1 (IDO1), indicating that these vaccines caused allergic disease suppression and reversal of respiratory tissue remodeling via generation of regulatory lymphocytes. Liposome entrapped-recombinant Per a 9 (L-Per a 9) did not cause upregulation of immunosuppressive cytokine genes and IDO1 increment; rather, L-Per a 9 induced high expression of IFN-γ in lungs of treated mice, which resulted in mitigation of allergic manifestations. This study provides compelling evidence that both liposome-entrapped vaccines made of single refined major allergen alone and single refined major allergen linked with Tregitopes are effective for reducing allergen-mediated respiratory tissue inflammation and remodeling, but through different mechanisms.

CD4 immunotherapy is potentially useful in immune reconstitution of CD4 + T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4 + T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4 + T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4 + T cells by immunorosette formation from healthy donors and cryopreserved CD4 + T cells from HIV-infected patients with CD4 count over 500 cells/μL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4 + T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4 + T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4 + T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4 + T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4 + T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: B cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection., Methods: In this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period., Results: Data shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs., Conclusions: Findings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. Dendritic cells (DCs) play a critical role in initiating and regulating highly effective antiviral immune response that include linking innate and adaptive immune responses. This study was conducted to comparatively characterize in detail the relative proportion, phenotypic changes, and maturation profile of subsets of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in children with dengue fever (DF), dengue hemorrhagic fever (DHF) and for purposes of control healthy individuals. The mDCs (Lin-CD11c+CD123lo), the pDCs (Lin-CD11c-CD123+) and the double negative (DN) subset (Lin-/HLA-DR+/CD11c-CD123-) were analyzed by polychromatic flow cytometry. The data were first analyzed on blood samples collected from DENV-infected patients at various times post-infection. Results showed that the relative proportion of mDCs were significantly decreased which was associated with an increase in disease severity in samples from DENV-infected patients. While there was no significant difference in the relative proportion of pDCs between healthy and DENV-infected patients, there was a marked increase in the DN subset. Analysis of the kinetics of changes of pDCs showed that there was an increase but only during the early febrile phase. Additionally, samples from patients during acute disease showed marked decreases in the relative proportion of CD141+ and CD16+ mDC subsets that were the major mDC subsets in healthy individuals. In addition, there was a significant decrease in the level of CD33-expressing mDCs in DENV patients. While the pDCs showed an up-regulation of maturation profile during acute DENV infection, the mDCs showed an alteration of maturation status. This study suggests that different relative proportion and phenotypic changes as well as alteration of maturation profile of DC subsets may play a critical role in the dengue pathogenesis and disease outcome., Competing Interests: The authors have declared that no competing interests exist.

Dengue virus (DENV) infection is considered one of the most important mosquito-borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV-infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56 hi CD16 - and CD56 lo CD16 + NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56 hi CD16 - subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56 lo CD16 + subset compared with those in the CD56 hi CD16 - subset. Moreover, although the CD56 lo CD16 + subset contained a high frequency of cells expressing skin-homing markers, the CD56 hi CD16 - subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection., (© 2017 John Wiley & Sons Ltd.)

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vβ region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor FcγRIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease., (Copyright © 2017, American Association for the Advancement of Science.)

Antiretroviral therapy (ART) is generally prescribed to patients with human immunodeficiency virus (HIV) infection with vaccination introduced to prevent disease complications. However, little is known about the influence of immunization on T cell subsets' distribution during the course of infection. This study aims to identify the impact of viral replication and immunization on naïve, effector, effector memory, and central memory T cell subpopulations in ART-treated HIV-infected children. Fifty patients were recruited and injected intramuscularly with influenza A (H1N1) 2009 vaccine on the day of enrollment (day 0) and day 28. Blood samples were collected for pre- and postvaccination on days 0 and 56 for analyzing T cell phenotypes by flow cytometry. Phenotypes of all T cell subsets remained the same after vaccination, except for a reduction in effector CD8 + T cells. Moreover, T cell subsets from patients with controllable viral load showed similar patterns to those with virological failure. Absolute CD4 count was also found to have a positive relationship with naïve CD4 + and CD8 + T cells. In conclusion, vaccination and viral replication have a little effect on the distribution of T cell subpopulations. The CD4 count can be used for prediction of naïve T cell level in HIV-infected patients responding to ART.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR + CD38 + and HLA-DR - CD38 + effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR + CD38 + subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue., Importance: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects., (Copyright © 2016 Chandele et al.)

Background: The activation of peripheral blood mononucleated cells (PBMCs) with anti-CD3/CD28-coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion., Objectives: The aim of this study was to define an optimal cell isolation protocol for the expansion of PBMCs using anti-CD3/CD28-coated bead stimulation, with the ultimate goal of using these cells for adoptive therapy., Material and Methods: PBMCs were isolated from healthy donor blood samples. To determine the effect of varying the bead-to-cell ratios on the expansion rate and phenotypic characterization of the expanded cells, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at bead-to-cell ratios of 0.1 : 1, 0.5 : 1 and 1.0 : 1 in the presence of 100 U/mL exogenous IL-2; also, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at a bead-to-cell ratio of 0.5 : 1 in the presence of varying concentrations of IL-2 (20, 100 and 1000 U/mL). Cell expansion was carried out for three weeks. The cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry., Results: The initial experiments showed no difference in the expansion rate from cells grown with the three different bead-to-cell ratios. PBMCs can be expanded up to 1000-fold at a 0.5 : 1 bead-to-cell ratio after three weeks of cell expansion with a high viability (90%). Furthermore, in the presence of 100 U/mL IL-2, the percentages of CD3-CD16+CD56+ cells showed marked increases., Conclusions: The results demonstrate that PBMCs were stimulated with anti-CD3/CD28-coated beads. This method may provide an alternative for driving T cell expansion, which may be very useful in adoptive immunotherapy.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations.

Unlabelled: Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis., Importance: In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived MAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However, in the case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the preexisting MBC pool. Our characterization of plasmablasts and plasmablast-derived MAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies provide evidence of OAS in the acute-phase dengue virus immune response, providing a basis for future work examining the impact of OAS phenotype antibodies on protective immunity and disease severity in secondary infections., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Designed molecular scaffolds have been proposed as alternative therapeutic agents against HIV-1. The ankyrin repeat protein (Ank(GAG)1D4) and the zinc finger protein (2LTRZFP) have recently been characterized as intracellular antivirals, but these molecules, used individually, do not completely block HIV-1 replication and propagation. The capsid-binder Ank(GAG)1D4, which inhibits HIV-1 assembly, does not prevent the genome integration of newly incoming viruses. 2LTRZFP, designed to target the 2-LTR-circle junction of HIV-1 cDNA and block HIV-1 integration, would have no antiviral effect on HIV-1-infected cells. However, simultaneous expression of these two molecules should combine the advantage of preventive and curative treatments. To test this hypothesis, the genes encoding the N-myristoylated Myr(+)Ank(GAG)1D4 protein and the 2LTRZFP were introduced into human T-cells, using a third-generation lentiviral vector. SupT1 cells stably expressing 2LTRZFP alone or with Myr(+)Ank(GAG)1D4 showed a complete resistance to HIV-1 in viral challenge. Administration of the Myr(+)Ank(GAG)1D4 vector to HIV-1-preinfected SupT1 cells resulted in a significant antiviral effect. Resistance to viral infection was also observed in primary human CD4+ T-cells stably expressing Myr(+)Ank(GAG)1D4, and challenged with HIV-1, SIVmac, or SHIV. Our data suggest that our two anti-HIV-1 molecular scaffold prototypes are promising antiviral agents for anti-HIV-1 gene therapy.

Aims: CD4+ T lymphocytes from HIV-infected patients with different degrees of disease progression based on CD4 count were expanded in vitro using anti-CD3/28-coated beads., Materials & Methods: Purified CD4+ T lymphocytes from healthy subjects and patients were expanded for 3 weeks. Moreover, the improvement of cell expansion by IL-2 supplementation was also determined., Results: Expanded CD4+ T lymphocytes from patients had lower fold expansion when compared with healthy subjects. Furthermore, patients with high CD4 counts had higher fold expansion than patients with low CD4 count, and IL-2 supplementation further increased cell expansion., Conclusions: Anti-CD3/28 activation failed to potently induce expansion of CD4+ T lymphocytes from patients. However, the cell expansion could be improved by IL-2 supplementation.

Dengue virus (DENV) infection induces the expansion of plasmablasts, which produce antibodies that can neutralize DENV but also enhance disease upon secondary infection with another DENV serotype. To understand how these immune responses are generated, we used a systems biological approach to analyze immune responses to dengue in humans. Transcriptomic analysis of whole blood revealed that genes encoding proinflammatory mediators and type I interferon-related proteins were associated with high DENV levels during initial symptomatic disease. Additionally, CD14(+)CD16(+) monocytes increased in the blood. Similarly, in a nonhuman primate model, DENV infection boosted CD14(+)CD16(+) monocyte numbers in the blood and lymph nodes. Upon DENV infection in vitro, monocytes upregulated CD16 and mediated differentiation of resting B cells to plasmablasts as well as immunoglobulin G (IgG) and IgM secretion. These findings provide a detailed picture of innate responses to dengue and highlight a role for CD14(+)CD16(+) monocytes in promoting plasmablast differentiation and anti-DENV antibody responses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dengue Virus (DV) infects between 50 and 100 million people globally, with public health costs totaling in the billions. It is the causative agent of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), vector-borne diseases that initially predominated in the tropics. Due to the expansion of its mosquito vector, Aedes spp., DV is increasingly becoming a global problem. Infected individuals may present with a wide spectrum of symptoms, spanning from a mild febrile to a life-threatening illness, which may include thrombocytopenia, leucopenia, hepatomegaly, hemorrhaging, plasma leakage and shock. Deciphering the underlining mechanisms responsible for these symptoms has been hindered by the limited availability of animal models that can induce classic human pathology. Currently, several permissive non-human primate (NHP) species and mouse breeds susceptible to adapted DV strains are available. Though virus replication occurs in these animals, none of them recapitulate the cardinal features of human symptomatology, with disease only occasionally observed in NHPs. Recently our group established a DV serotype 2 intravenous infection model with the Indian rhesus macaque, which reliably produced cutaneous hemorrhages after primary virus exposure. Further manipulation of experimental parameters (virus strain, immune cell expansion, depletion, etc.) can refine this model and expand its relevance to human DF. Future goals include applying this model to elucidate the role of pre-existing immunity upon secondary infection and immunopathogenesis. Of note, virus titers in primates in vivo and in vitro, even with our model, have been consistently 1000-fold lower than those found in humans. We submit that an improved model, capable of demonstrating severe pathogenesis may only be achieved with higher virus loads. Nonetheless, our DV coagulopathy disease model is valuable for the study of select pathomechanisms and testing DV drug and vaccine candidates.

Backgrounds: Activation of CD4+ T lymphocytes with anti-CD3/CD28 coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. The propose of this study is to define the optimal cell isolation protocol for expansion of CD4+ T lymphocytes by using anti-CD3/CD28 coated bead stimulation with an ultimate goal of using these cells for adoptive immunotherapy., Methods: CD4+ T cells were isolated from healthy donor blood samples using three different methods including immunorosette formation, negative selection and CD8 depletion using immunomagnetic beads. These cells were activated with anti-CD3/CD28 coated beads at a bead to cell ratio of 1:1 and cell expansion was carried for 3 weeks. Cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry., Results: Purified CD4+ T lymphocytes which were isolated via immunorosette formation can be expanded up to 1000-fold within 3 weeks with high viability (90%and high purity of CD4+ T lymphocytes (>95%). However, cell expansion from purified CD4+ T lymphocytes which were isolated by negative selection and CD8-depletion provided approximately 300-fold expansion., Conclusions: The results demonstrate that purified CD4+ T lymphocytes from immunorosette formation provided the highest CD4+ T lymphocyte expansion when stimulated with anti-CD3/CD28 coated beads. This method can be used to obtain a large number of expanded CD4+ T cells for adoptive immunotherapy.

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4(+) Tregs, CD8(+) Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4(+) Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8(+) Tregs was also noted with a moderate increase in ECs but further increases of CD8(+) Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4(+) Tregs and increases in the levels of CD8(+) Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17(+) subsets in GIT as CD3(-/)CD8(+)/NKG2a(-) and CD3(+)/CD8(+)/NKG2a(+) subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design.

Immunization with a pandemic influenza A H1N1 2009 was recommended for HIV-infected patients. However, there is limited information concerning the impact of immunization with this vaccine on immune activation and HIV viral replication. In this study, 45 HIV-infected children and adolescents receiving antiretroviral therapy were immunized with a 2-dose series of nonadjuvated monovalent influenza A H1N1 2009 vaccine upon enrollment and approximately 1 month later. Immunogenicity was determined by haemagglutination inhibition assay. The level of immune activation was determined by identification of CD38 and HLA-DR on CD8+ T cells. Patients were divided into 2 groups which include patients who had an undetectable HIV viral load (HIV detectable group) and patients who show virological failure (HIV nondetectable group). The results showed seroconversion rate of 55.2% in HIV nondetectable group, whereas 31.3% was found in HIV detectable group. Both groups of patients showed no major increase in immune activation after immunization. Interestingly, a decrease in the frequency of CD8+ T cells that coexpressed CD38 and HLA-DR was observed after immunization in both groups of patients. We suggested that immunization with influenza A H1N1 2009 vaccine can induce immune response to the pandemic virus without major impact on HIV viral replication and immune activation.

Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41(+)CD61(+) cells, regardless of CD34 and CD45 expression. The CD61(+) cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting-assisted isolation of CD61(+) cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. All rights reserved.)

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

Background: Although the impairment of HIV-specific T lymphocytes is evident during chronic HIV-infection, it is unclear whether the increased CD8+ T cells associates with either selective or overall change of effector functional phenotype. Instead of study on HIV-specific T cells only, analyzing bulk T cell populations represent a neglected area of T cell impairment, which go far beyond HIV-specific T cells., Methods: In this study, we determined the diversity of CD8+ T cells in term of cytolytic molecule expression (perforin, granzyme A, and granzyme B) and cytokine production ability (IFN-gamma, TNF-alpha, and IL-2) using intracellular staining and flow cytometry technique. The results were compared between healthy individuals, untreated, and antiretroviral therapy (ART) treated HIV infected patients., Results: We demonstrated the presence of four different subsets of CD8+ T cells that expressed different combinations of cytolytic molecules. We also identified seven different subsets of cytokine producing cells based on different combination of IFN-gamma, TNF-alpha, and IL-2. Results showed significant alterations of these cell subsets that expressed different combination of cytolytic effector molecules or cytokines in HIV infected patients. Furthermore, cytolytic molecule expressing cell subsets are not normalized in effective ART treated patients, whereas the selective population of cytokine producing cells returned to normal value., Conclusions: The effector diversity of CD8+ T cells changed in HIV infected patients. Although effective ART altered functional diversity of these cells, long-term suppression of viral replication may be required to normalize the selected CD8+ T cell effector phenotype in HIV infected patients., (Copyright © 2011 International Clinical Cytometry Society.)

Background: Enumeration of CD4+ T-lymphocytes is important in the management of HIV. However, standard laboratory systems based on flow cytometry are expensive, complicated, and thus unavailable to most resource-limited settings where a low-cost and fully automated point-of-care CD4 testing system is required. In attempts to address this issue, a study was conducted to validate the Alere PIMA point-of-care CD4 test., Method: Duplicate values of the absolute number of CD4+ T-lymphocytes in 203 HIV-infected blood samples obtained using the PIMA system were compared with the two predicate single-platform FACSCount and the dual-platform FACSCan (Becton Dickinson Biosciences)., Results: The overall absolute CD4+ T-lymphocyte count obtained using the PIMA system correlated highly with the FACSCount (r = 0.957; mean bias, -54.2 cells/μL; limit of agreement, -190.9 to +82.5 cells/μL) and the FACSCan (r = 0.957; mean bias -44.0 cells/μL; limit of agreement, -179.7 to +91.6 cells/μL). Good correlation and low biases were also observed for samples with CD4+ T-lymphocyte count ranges of 0 to 200 and 0 to 350 cells/μL. Additionally, there was no significant difference in absolute CD4+ T-lymphocyte counts noted between the duplicate samples using the PIMA system., Conclusions: This new point-of-care product is a simple and reliable system and should contribute significantly to the simplification of performing CD4 testing and thus increase access for patients in resource-limited settings. The inability to obtain values for the frequency (%) of CD4+ T-lymphocyte count is one limitation of the PIMA system, the addition of which would be of value for clinical staging or monitoring in HIV-infected pediatric patients.

Background: The frequency and absolute number of CD4+ T-lymphocytes continue to be one of the major clinical markers for management of HIV/AIDS. The present standard dual-platform (DP) three-color and two-color PanLeucogating flow cytometric (FCM) methods for most developing countries are either expensive if manufacturers' monoclonal antibody reagents are used or limited due to an insufficient supply of generic reagents. Clearly, more affordable FCM methods are needed., Objective: To develop a novel DP FCM method using biotin-streptavidin-fluorochrome labeling in combination with the two standard DP methods for 4 different white blood cells (WBC) using only one monoclonal antibody reagent., Methods: The percentage of CD4+ T-lymphocytes in 116 HIV-infected blood samples was determined using our new method. Results were compared with the two standard methods. Correlation and agreement of the pair method were determined using linear regression, Bland-Altman and percent similarity analysis., Results: Our study showed that percentage of CD4+ T-lymphocyte values obtained from the new method correlated highly with the standard three-color and the two-color methods (r2 = 0.95 {n=52} and 0.97 {n=64}). The mean bias and percent similarity for the new method compared with the two standard methods were -0.53% (limit of agreement {LOA}:-5.22% to +4.16% with percent similarity of 99.28; and -0.22% with LOA of -3.42% to +2.98%, the percent similarity of 98.15, respectively., Conclusions: Our FCM method using biotin to label 4 different WBC samples followed by streptavidin staining is reliable for determination of CD4+ T-lymphocytes. Such an approach will significantly reduce the cost for monitoring HIV-infected patients in resource-limited settings.

Background: We have previously shown that monitoring of CD38 expression can be used as a marker for antiretroviral drug efficacy in HIV infected patients. However, the detection of CD38 expression may be affected by the sensitivity of the fluorochrome conjugated reagent., Objective: In this study, we determined the level of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies in different groups of HIV infected patients., Methods: The frequency and mean fluorescence intensity of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies were detected by flow cytometry either alone or in combination with HLA-DR. A correlation between CD38 expression and CD4 count, the percentage of CD4 or viral load in antiretroviral drug naive HIV infected patients was performed. The results were compared with those for antiretroviral treated HIV infected patients who responsed to therapy and patients with virological failure., Results: We found that while both reagents had the ability to detect a high frequency of CD38 expressing cells in untreated patients, only PE conjugated reagent provided correlation with markers for disease progression. More importantly, FITC conjugated reagent cannot monitor the increase in CD38 expression in patients who showed virological failure., Conclusions: The results from this study suggest that a cautious selection of fluorochrome conjugated reagents and a method for utilizing the data are extremely critical in the use of CD38 expression as a monitoring tool for ART efficacy.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

Although XMRV dissemination in humans is a matter of debate, the prostate of select patients seem to harbor XMRV, which raises questions about its potential route of transmission. We established a model of infection in rhesus macaques inoculated with XMRV. In spite of the intravenous inoculation, all infected macaques exhibited readily detectable XMRV signal in the reproductive tract of all 4 males and 1 female during both acute and chronic infection stages. XMRV showed explosive growth in the acini of prostate during acute but not chronic infection. In seminal vesicles, epididymis, and testes, XMRV protein production was detected throughout infection in interstitial or epithelial cells. In the female monkey, epithelial cells in the cervix and vagina were also positive for XMRV gag. The ready detection of XMRV in the reproductive tract of male and female macaques infected intravenously suggests the potential for sexual transmission for XMRV.

Lack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype 2 (strain 16 681) at 1 x 10(7) PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of D-dimer was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. Dengue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.

CD4(+) T-lymphocyte count is an important marker in management of HIV-1-infected patients. The standard single-platform (SP) flow cytometric (FCM) CD4(+) testing that uses the known reference microbeads is expensive; more affordable alternatives are therefore needed. We evaluated the use of glutaraldehyde-fixed chicken red blood cells (CRBCs) as counting beads as an alternative for enumerating CD4(+) T-lymphocyte counts in 87 HIV-1-infected patients. Linear regression analyses revealed an excellent correlation of the SP FCM using CRBCs with the standard SP bead-based FCM method (percentages, r(2) > 0.99; absolute counts, r(2) > 0.98) over the entire range including the clinically relevant range. Mean percent bias for the CRBC method was +0.35% [limits of agreement (LOA): -1.86% to +2.57%]. For absolute CD4(+) T-lymphocytes, the mean biases was -47.76 cells per microliter (LOA: -191.34 to +98.81 cells/microL) with much lower bias for CD4 T-lymphocyte counts <200 cells per microliter (LOA: -31.92 to +22.95 cells/microL). The use of CRBCs is comparable with the use of commercial microbeads. This has resulted in major cost savings to resource-limited countries where the health care system is under increasing pressure to operate cost effectively. This can greatly facilitate and ensure the success of the ongoing antiretroviral therapy program in these countries.

Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

Viremia is one of the features of dengue virus infection among the flaviviruses. Dengue virus infection results in a spectrum of clinical symptoms, ranging from undifferentiated flu-like illness, mild dengue fever, to dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a life-threatening illness. Several mechanisms have been hypothesized based primarily on data collected from post-acute clinical phase to account for DHF/DSS. Lack of a suitable animal model for DHF/DSS has hindered progress in defining the etiology of DHF/DSS. Levels of circulating dengue virus have been well-correlated to severe dengue disease. However, the cell lineage(s) serving as a primary target for the source of viremia are largely unknown. Results from in vivo and in vitro pilot studies using molecular and more advanced technologies reveal that dengue virus appears to be associated with platelets and the megakaryocytic lineage. The observation may partially explain the dysfunction of platelets observed in dengue affected patients.